Sincalide Formulations

ABSTRACT

The invention features sincalide formulations that include an effective amount of sincalide, a bulking agent/tonicity adjuster, a stabilizer, a surfactant, a chelator, and a buffer. The invention also features kits and methods for preparing improved sincalide formulations as well as methods for treating, preventing, and diagnosting gall bladder-related disorders using sincalide formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 12/607,337, filed Oct. 28, 2009, which is a continuation ofco-pending U.S. patent application Ser. No. 10/921,732, filed Aug. 19,2004, now abandoned, and which is a continuation of and claims priorityto U.S. patent application Ser. No. 10/222,540, filed Aug. 16, 2002, nowgranted as U.S. Pat. No. 6,803,046, all of which are hereby incorporatedby reference.

FIELD OF THE INVENTION

The invention relates to pharmaceutically acceptable formulations ofsincalide.

BACKGROUND OF THE INVENTION

KINEVAC® (Sincalide for Injection, USPe) is acholecystopancreatic-gastrointestinal hormone peptide for parenteraladministration. The active pharmaceutical ingredient,1-De(5-oxo-L-glutamine-5-L-proline)-2-de-L-methioninecaerulein or“sincalide” (CAS#25126-32-3), is a synthetically prepared C-terminaloctapeptide of cholecystokinin (CCK-8), with the following amino acidsequence: Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂.

KINEVAC® was first introduced in 1976, and was finished as a sterile,nonpyrogenic, lyophilized white powder in a 5-mL (nominal) glass vial tocontain: 5 μg sincalide with 45 mg sodium chloride to provide tonicity;sodium hydroxide or hydrochloric acid may have been added for pHadjustment (pH 5.5-6.5). The type I glass vial was sealed under anitrogen headspace with a Tompkins B0849 closure. This two-ingredientformulation was incorporated into the U.S. Pharmacopea/NationalFormulary, USP 24, NF 19, Jan. 1, 2000.

Since its introduction, various drawbacks in the manufacturing andanalysis of KINEVAC® have been identified. For example, thetwo-ingredient formulation suffers from potency variability. Thisvariability was exacerbated by the fact that the formulation wasanalyzed using a guinea pig gallbladder contraction bioassay for potencyof both sincalide and KINEVAC®. This bioassay was unable to distinguishbetween bioactivity of sincalide and bioactivity of sincalidedegradants. Accordingly, a 20% overage of sincalide was required inprevious sincalide formulations to compensate for the limitations of thebioassay. Thus, there is a need for sincalide formulations havingimproved and consistent potency as established by a sincalide specificassay such as HPLC.

SUMMARY OF THE INVENTION

The present invention satisfies the need for improved sincalideformulations by providing formulations that eliminate the need for a 20%overage of sincalide. The sincalide formulations of the invention arealso purer than prior art formulations, and have fewer degradants andmore consistent potency. In addition, the purity of these formulationsmay be assessed by HPLC, thus eliminating the need for the bioassay ofthe prior art formulations.

The present invention provides sincalide formulations adapted foradministration by injection. These sincalide formulations arecharacterized by improved stability and may be prepared as a relativelylarge volume batch (≈100 L).

In one aspect, the invention features sincalide formulations thatinclude an effective amount of sincalide, a bulking agent/tonicityadjuster, one or more stabilizers, a surfactant, a chelator, and abuffer. The invention also features kits and methods for preparingimproved sincalide formulations, as well as methods for treating,preventing, and diagnosing gall bladder-related disorders usingsincalide formulations.

The formulations of the invention preferably have a pH between 6.0 and8.0. Suitable buffers include, but are not limited to, phosphate,citrate, sulfosalicylate, borate, acetate and amino acid buffers.Phosphate buffers, such as dibasic potassium phosphate, are preferred.

In various embodiments of the invention, the surfactant is a nonionicsurfactant, preferably a polysorbate, such as polysorbate 20 orpolysorbate 80; the chelator is pentetic acid (DTPA); and the stabilizeris an antioxidant and/or amino acid. In a particularly desirableembodiment of the invention, the formulation includes a plurality ofstabilizers, preferably L-arginine monohydrochloride, L-methionine,L-lysine monohydrochloride, and sodium metabisulfite.

Suitable bulking agents/tonicity adjusters include, but are not limitedto, mannitol, lactose, sodium chloride, maltose, sucrose, PEG's,cyclodextrins, dextran, polysucrose (Ficoll), and polyvinylpyrrolidine(PVP). D-Mannitol is a preferred bulking agent/tonicity adjuster.

In a particularly preferred embodiment, the reconstituted formulationincludes 0.0008 to 0.0012 mg/mL active ingredient (i.e., sincalide);20.0 to 50.0 mg/mL mannitol, 2.0 to 7.0 mg/mL arginine; 0.2 to 1.0 mg/mLmethionine; 2.0 to 30.0 mg/mL lysine; 0.002 to 0.012 mg/mL sodiummetabisulfite; 0.000001 to 0.003 mg/mL polysorbate 20, 0.1 to 3.0 mg/mLpentetic acid (DTPA); and 5.4 to 12.0 mg/mL potassium phosphate(dibasic). In a more preferred embodiment, the reconstituted formulationincludes about 0.001 mg/mL sincalide; about 34 mg/mL D-mannitol, about 6mg/mL L-arginine monohydrochloride; about 0.8 mg/mL L-methionine; about3 mg/mL L-lysine monohydrochloride; about 0.008 mg/mL sodiummetabisulfite; less than about 0.01 mg/mL polysorbate 20, about 0.4mg/mL pentetic acid (DTPA); and about 1.8 mg/mL potassium phosphate(dibasic).

The kits of the invention may, for example, include the variouscomponents of the formulation as a mixture in powder form, along with acontainer (e.g., a vial) to hold the powder mixture and aphysiologically acceptable fluid for reconstitution of the formulation.The components of the formulation may be present in the kit either inthe powder mixture or in the fluid portion. Kits of the invention mayalso include all components in a liquid mixture or some components in aliquid form and some in the form of a powder.

The formulations of the invention have improved stability and potencycompared to previous sincalide formulations, and are useful asdiagnostic aids for imaging the hepatobiliary system of a patient. Whenused as a diagnostic aid, the sincalide formulations may, for example,be co-administered with a radiopharmaceutical agent having rapid hepaticuptake, such as ^(99m)Tc-mebrofenin, or similar hepatobiliary imagingagents, to assist in the diagnosis of gallbladder diseases and relateddisorders. Additionally, the formulations may be administered beforeand/or after diagnostic imaging (including for example, magneticresonance imaging, scintigraphic imaging, ultrasound imaging, etc.)

The sincalide formulations of the invention may also be administered topatients receiving total parenteral nutrition (TPN), in order to treatand/or prevent TPN-related disorders.

Other features and advantages of the invention will be apparent from thefollowing detailed description thereof and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing illustrating the chemical structure of1-De(5-oxo-L-glutamine-5-L-methioninecaerulein or “sincalide”(CAS#25126-32-3). The amino acid residues “Met 3” and “Met 6” areoutlined by dashed lines.

FIG. 2 is a drawing illustrating the chemical structure of sincalide(Met 3) monosulfoxide.

FIG. 3 is a drawing illustrating the chemical structure of sincalide(Met 6) monosulfoxide.

FIG. 4 is a drawing illustrating the chemical structure of sincalide(Met 3, 6) disulfoxide.

FIG. 5 is a graphical representation of the effect of pH on the recoveryof sincalide in 35 mM phosphate buffer over 24 hours. At each pH forwhich data is shown, the bars represent 0, 6, and 24 hours, from left toright.

FIG. 6 is a graphical representation of the effect of pH on the recoveryof sincalide in a formulation of the invention over 8 hours. At each pHfor which data is shown, the bars represent 0, 4, and 8 hours, from leftto right.

FIG. 7 is a graphical representation of the percent sincalide Met 3 andMet 6 monosulfoxides (vs sincalide), in the presence and absence ofpentetic acid (DTPA).

FIG. 8 is a chromatogram of KINEVAC® experimental formulation (no DTPA)spiked with 0.63 mM Cu²⁺.

FIG. 9 is a chromatogram of KINEVAC® experimental formulation (1 mMDTPA) spiked with 0.63 mM Cu²⁺.

FIG. 10 is a chromatogram of KINEVAC® experimental formulation (no DTPA)spiked with 0.18 mM Mn²⁺.

FIG. 11 is a chromatogram of KINEVAC® experimental formulation (1 mMDTPA) spiked with 0.18 mM Mn²⁺.

FIG. 12 shows representative full-scale and expanded scale chromatogramsof a lyophilized reformulation of KINEVAC® upon reconstitution with 5 mLwater, resulting in a sincalide concentration of 1 μg/mL.

DETAILED DESCRIPTION OF THE INVENTION

In order to develop an improved sincalide formulation a series ofstudies, described in the Examples below, were conducted to determinethe effects of various excipients on formulations of sincalide. Throughthese studies, we discovered that the potency and stability of sincalideformulations can be significantly enhanced through the careful selectionof excipients that provide certain desired functions. Accordingly, thepresent invention provides novel sincalide formulations having improvedstability and/or potency over previous formulations.

As used herein, the term “sincalide” includes the synthetically-preparedC-terminal octapeptide of cholecystokinin (CCK-8), with the amino acidsequence: Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, as well asderivatives thereof which have been optimized or modified (to improvestability, potency, pharmacokinetics, etc.), but retain the biologicalactivity of the original octapeptide. For example, peptides in which themethionine and/or aspartic acid residues have been replaced withoutsignificantly affecting the biological activity are included within“sincalide” as the term is used herein. Similarly, the term “sincalide”encompasses not only monomeric, but multimeric forms of the peptide, aswell as physiologically active degradants or portions of the peptide andits derivatives.

The sincalide formulations of the invention can include a variety ofexcipients, such as, for example, antioxidants, buffers, bulkingagents/tonicity adjusters, chelating agents, complexing agents,crosslinking agents, co-solvents, osmolality adjustors, solubilizers,surfactants, stabilizers, pH adjustors, lyoprotectants/cryoprotectants,air/liquid and/or ice-liquid interface protectants (protectants againstsurface induced denaturation), freeze-thaw protectants, protectantsagainst protein/peptide denaturation, protectants for rehydration, andwetting agents. In preferred embodiments, the formulations includeexcipients that perform the functions of at least: (i) a bulkingagent/tonicity adjuster, (ii) a stabilizer, (iii) a surfactant, (iv) achelator, and (v) a buffer. Typically, each of these functions isperformed by a different excipient. However, in some embodiments of theinvention a single excipient may perform more than one function. Forexample, a single excipient may be multi-functional, e.g. amino acidsmay function as bulking agents, stabilizers and/or buffers and otherexcipients may function, for example, as both a stabilizer and achelator or as both a bulking agent and a tonicity adjuster.Alternatively, multiple excipients serving the same function may beused. For example, the formulation may contain more than one excipientthat functions as a stabilizer.

Table 1 below shows the concentration ranges for various excipients thatwere investigated. In general, the range studies were based on a 2-mLfill of bulk solution per vial before lyophilization. Afterreconstitution with 5 mL of water for injection the final sincalideformulation results in an isotonic solution. The concentration ranges ofthe various ingredients provided in Table 1 can be adjusted upward ordownward, if necessary in conjunction with: increasing or decreasing thefill volume per vial, obtaining the desired pH, obtaining the desiredreconstitution volume, and the desirability of achieving tonicity in thefinal reconstituted solution. For example, as indicated above, theconcentrations provided in Table 1 were developed to provide an isotonicsolution; however, one skilled in the art would recognize that a broaderrange of concentrations could be used if an isotonic solution was notrequired.

TABLE 1 Concentration ranges for excipients for preferred sincalideformulations. Final Formulation (mg) Range 1 mL Range Range (mg per 1 mL1 mL 1 vial after Excipient Function (mg/mL Bulk) (mg/vial) afterreconst) Bulk Target reconst. (Sincalide) Active Ingredient 0.00250.0050 0.0008-0.0012 0.0025 0.0050 0.0010 Mannitol Bulking Agent/ 50.0-125.0 100-250 20.0-50.0 85 170 34 Cake Forming Agent/ TonicityAdjuster TWEEN ®-20 Non-Ionic Surfactant/ 0.0000025-0.0075  0.0000050-0.0150   0.0000010-0.0030   <0.01 <0.01 <0.01 SolubilizingAgent/ Wetting Agent DTPA Chelator/Stabilizer/ 1.0   2.0   0.1-3.0 1.02.0 0.4 Antioxidant/ Complexing Agent/ Preservative/pH Adjuster SodiumAntioxidant/Preservative/ 0.005-0.030 0.010-0.060 0.002-0.012 0.0200.040 0.008 Metabisulfite Stabilizer Potassium Buffer/pH Adjuster/2.7-4.5  5.4-12.0 1.1-1.8 4.5 9.0 1.8 Phosphate, Dissolution Aid dibasicPotassium Buffer/pH Adjuster/ 1.0-6.5  9.6-13.0 1.92-2.6  0 0 0Phosphate, Dissolution Aid monobasic Methionine Stabilizer 0.5-2.51.0-5.0 0.2-1.0 2.0 4.0 0.8 Lysine Stabilizer/Lyoprotectant/  5.0-30.010.0-60.0  2.0-30.0 7.5 15.0 3.0 Cryoprotectant ArginineStabilizer/Lyoprotectant/  5.0-17.5 10.0-35.0 2.0-7.0 15 30.0 6.0Cryoprotectant/pH Adjuster Sodium Tonicity Adjuster 4.5-9.0  9.0-18.01.8-3.6 0 0 0 Chloride Alternative excipients include TWEEN ®-80,potassium metabisulfite, sodium phosphate dibasic, sodium phosphatemonobasic, and potassium chloride. Additional alternatives are listedbelow.

Table 2 shows preferred ranges for preferred excipients in the bulksolutions, vials and after reconstitution. All concentrations shown forthe bulk solution are based on a 2 mL fill volume. The ingredientquantities are matched to result in a pH slightly below neutral andresult in an isotonic solution after reconstitution of the lyophilizedvial as indicated by an osmolality in the range of 180 to 320 mOsm/kg,preferably, 240 to 320 mOsm. The columns titled “Final Formulation”represent particularly preferred formulations.

TABLE 2 Osmolality values for various sincalide formulations. (Allformulations contain 0.0025 mg CCK-8/mL.; “dibasic” and “monobasic”refer to dibasic and monobasic potassium phosphate; “Na meta” refers tosodium metabisulfite) Formulation Excipients Calculated (mg/mL Bulk)mOsm/kg Mannitol (125.0) 292 Dibasic (3.75) DTPA (1.0) Mannitol (95.0)244 Dibasic (4.0) Monobasic (2.8) DTPA(1.0) Mannitol (103.0) 244 Dibasic(3.75) DTPA (1.0) Mannitol (75.0) 244 NaCl (4.5) Dibasic (3.75) DTPA(1.0) Mannitol (85.0) 187 TWEEN ® 20 (0.005) Dibasic (2.75) DTPA (1.0)Methionine (2.0) Lysine (15.0) Mannitol (50.0) 247 NaCl (9.0) Dibasic(3.00) DTPA (1.0) TWEEN ® 20 (0.0075) 264 Mannitol (75.0) KCl (6.0)Dibasic (3.25) Monobasic (1.0) DTPA (1.0) Methionine (2.0) TWEEN ® 20(0.005) 264 Mannitol (75.0) KCl (6.0) Dibasic (3.25) Monobasic (1.0)DTPA (1.0) Methionine (2.0) TWEEN ® 20 (0.0025) 264 Mannitol (75.0) KCl(6.0) Dibasic (3.25) Monobasic (1.0) DTPA (1.0) Methionine (2.0) TWEEN ®20 (2.5 ng) 314 Mannitol (85.0) Dibasic (4.50) DTPA (1.0) Nametabisulfite (0.020) Methionine (2.0) Lysine (7.50) Arginine (15.0) NaMeta (0.015) 257 Mannitol (85.0) Dibasic (2.75) DTPA (1.0) 20 (0.005)Methionine (2.0) Lysine (7.50) Arginine (15.0) Na Meta (0.030) 257Mannitol (85.0) Dibasic (2.75) DTPA (1.0) TWEEN ® 20 (0.005) Methionine(2.0) Lysine (7.50) Arginine (15.0) Na Meta (0.005) 257 Mannitol (85.0)Dibasic (2.75) DTPA (1.0) TWEEN ® 20 (0.005) Methionine (2.0) Lysine(7.50) Arginine (15.0) Na Meta (0.020) 259 Mannitol (85.0) Dibasic(3.00) DTPA (1.0) TWEEN ® 20 (0.005) Methionine (2.0) Lysine (7.50)Arginine (15.0) Dibasic (2.75) 257 Mannitol (85.0) Na Meta (0.015) DTPA(1.0) TWEEN ® 20 (0.005) Methionine (2.0) Lysine (7.50) Arginine (15.0)Dibasic (3.00) 259 Mannitol (85.0) Na Meta (0.020) DTPA (1.0) TWEEN ® 20(0.005) Methionine (2.0) Lysine (7.50) Arginine (15.0) Dibasic (3.25)264 Mannitol (75.0) KCl (6.0) TWEEN ® 20 (0.0025) Monobasic (1.0) DTPA(1.0) Methionine (2.0) Dibasic (4.50) 314 Mannitol (85.0) TWEEN ® 20(2.5 ng) DTPA (1.0) Na metabisulfite (0.020) Methionine (2.0) Lysine(7.50) Arginine (15.0) Methionine (2.0) 262 Mannitol (75.0) NaCl (5.0)TWEEN ® 80 (0.025) Monobasic (1.0) DTPA (1.0) Dibasic (3.25) Methionine(1.5) 262 Mannitol (75.0) NaCl (5.0) TWEEN ® 80 (0.025) Monobasic (1.0)DTPA (1.0) Dibasic (3.25) Methionine (1.0) 262 Mannitol (75.0) NaCl(5.0) TWEEN ® 80 (0.025) Monobasic (1.0) DTPA (1.0) Dibasic (3.25)Methionine (0.5) 262 Mannitol (75.0) NaCl (5.0) TWEEN ® 80 (0.025)Monobasic (1.0) DTPA (1.0) Dibasic (3.25) Methionine (2.5) 262 Mannitol(75.0) NaCl (5.0) TWEEN ® 80 (0.005) Monobasic (1.0) DTPA (1.0) Dibasic(3.25) Lysine (5.0) 209 Mannitol (95.0) TWEEN ® 20 (0.005) Dibasic(2.75) DTPA (1.0) Methionine (2.0) Lysine (15.0) 187 Mannitol (85.0)TWEEN ® 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0) Lysine(30.0) 245 Mannitol (70.0) TWEEN ® 20 (0.005) Dibasic (2.75) DTPA (1.0)Methionine (2.0) Arginine (17.5) 245 Mannitol (85.0) TWEEN ® 20 (0.005)Dibasic (2.75) DTPA (1.0) Methionine (2.0) Arginine (10.0) 232 Mannitol(85.0) TWEEN ® 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0)Arginine (5.0) 238 Mannitol (85.0) TWEEN ® 20 (0.005) Dibasic (2.75)DTPA (1.0) Methionine (2.0) Lysine (7.5) Arginine (8.75) 245 Mannitol(85.0) TWEEN ® 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0)Lysine (7.5) Arginine (15.0) 257 Mannitol (85.0) TWEEN ® 20 (0.005)Dibasic (2.75) DTPA (1.0) Methionine (2.0) Lysine (7.5)

Chelators

Excipient impurities and/or stopper extractables can introduce tracemetals into pharmaceutical formulations. Sincalide contains twomethionine residues (Met 3 and Met 6) that are susceptible to oxidationby free metals. Thus, the sincalide formulations of the inventioncontain chelators to inhibit the oxidation of the two methionineresidues present in sincalide (Met 3 and Met 6). Preferred chelatorsinclude pentetic acid (DTPA), edetic acid (EDTA) and derivativesthereof, including salts. DTPA is a preferred chelator. As described inExample 2 below, the amounts of the degradants, sincalide Met 3 andsincalide Met 6 monosulfoxides, increase in the presence of certainmetals and in the absence of DTPA, while the presence of DPTA has aninhibitory effect on the formation of these monosulfoxides. Inparticular, copper and manganese, in the absence of DTPA, have thegreatest oxidative effect on the methionine residues of sincalideresulting in combined height percentages of Met 3 and Met 6monosulfoxides (vs sincalide) of 85.5 and 128.9, respectively.

In a preferred embodiment, the sincalide formulations contain between0.1 and 3.0 mg of DTPA per mL after reconstitution. In a particularlypreferred embodiment, sincalide formulations of the invention contain0.4 mg DTPA/mL after reconstitution with 5 mL.

Buffering Agents

Buffering agents are employed to stabilize the pH of sincalideformulations of the invention, and consequently, reduce the risk ofchemical stability at extreme pH values. Buffering agents useful in thepreparation of formulation kits of the invention include, but are notlimited to, phosphoric acid, phosphate (e.g. monobasic or dibasic sodiumphosphate, monobasic or dibasic potassium phosphate, etc.), citric acid,citrate (e.g. sodium citrate, etc.), sulfosalicylate, acetic acid,acetate (e.g. potassium acetate, sodium acetate, etc.), methyl boronicacid, boronate, disodium succinate hexahydrate, amino acids, includingamino acid salts (such as histidine, glycine, lysine, imidazole), lacticacid, lactate (e.g. sodium lactate, etc.), maleic acid, maleate,potassium chloride, benzoic acid, sodium benzoate, carbonic acid,carbonate (e.g. sodium carbonate, etc.), bicarbonate (e.g. sodiumbicarbonate, etc.), boric acid, sodium borate, sodium chloride, succinicacid, succinate (e.g. sodium succinate), tartaric acid, tartrate (e.g.sodium tartrate, etc.), tris-(hydroxymethyl)aminomethane, biologicalbuffers (such as N-2-hydroxyethylpiperazine,N′-2-ethanesulfonic acid(HEPES), CHAPS and other “Good's” buffers), and the like.

Phosphate is a preferred buffering agent due to its lack of interactionwith sincalide and an ideal buffering capacity in the physiological pHrange. Dibasic potassium phosphate is a particularly preferred buffer insincalide formulations of the invention. As described in Example 1below, a sincalide formulation of the invention proved to be stable overa pH range of 5.5-9.1. Within the pH range of 5.5-8.5, no distinctpH-dependent related trends in initial sincalide recovery were observedwith a sincalide formulation of the invention. Preferably, a sincalideformulation of the invention has a pH from 6.0 to 8.0.

Stabilizers

The octapeptide, sincalide, contains one tryptophan and two methionineresidues. Methionine has been identified as one of the most easilyoxidizable amino acids, which degrades to its corresponding sulfoxideand, under more strenuous oxidation conditions, its sulfone. Themechanisms of oxidation appear to be highly dependent on the reactiveoxygen species under consideration: peroxide, peroxyl radicals, singletoxygen, and hydroxyl radical have all been shown to oxidize methionineresidues to sulfoxides and other products. Therefore, based on thepotential for oxidation of this peptide, it was necessary to identifyfunctional additives for peptide stabilization.

Antioxidants/Reducing Agents.

In a preferred embodiment of the invention, the sincalide formulationcontains an antioxidant or reducing agent as a stabilizer. A widevariety of antioxidants or reducing agents can be used as stabilizers,including but not limited to, acetylcysteine, cysteine, ascorbic acid,benzyl alcohol, citric acid, pentetic acid or diethylenetriaminepentaacetic acid (DTPA), propyl gallate, methylparaben, sulfoxylate,propylparaben, edetic acid or ethylenediaminetetraacetic acid (EDTA),disodium EDTA dihydrate, dithiothreitol, glutathione, monothioglycerol,potassium metabisulfite, sodium formaldehyde sulfoxylate, sodiumsulfite, sodium succinate, sodium metabisulfite, stannous chloride,thioacetic acid, thiodiglycerol, thioethanolamine, thioglycolic acid,2-aminoethanethiol (cysteamine), butylated hydroxyanisole (BHT), andsodium sulfate and derivatives thereof, including salts and sulfurousacid salts. Sodium metabisulfite is a preferred antioxidant stabilizer.Additionally, DTPA, which is a preferred chelator, also may be anantioxidant stabilizer.

Amino Acids.

Amino acids have also been used as stabilizers or co-stabilizers ofpeptides to: act as cryoprotectants during freeze drying, stabilizeagainst heat denaturation, inhibit aggregate formation, improvesolubility or rehydration, inhibit isomerization, reduce surfaceadsorption, or act as chelating agents. They can also increase theproduct glass transition temperature (T_(g)) and thereby increaseprocess stability, as well as stabilize the product by minimizingoverdrying during secondary drying. Surface exposed residues can reactreadily with oxidizing agents at physiological pH, scavenging oxidizingmolecules and protecting critical regions of peptides.

Various D- and/or L-amino acids can be used as stabilizers in sincalideformulations. As used herein “amino acid(s)” and the names of specificamino acids (e.g arginine, lysine, methionine, etc.) encompass D- and/orL-amino acids, amino acid salts, derivatives, homologs, dimers,oligomers, or mixtures thereof. Preferred amino acids for use asstabilizers in the present invention include methionine, lysine, andarginine. Examples of other amino acids (and amino acid salts) suitableas stabilizers include, but are not limited to, arginine glutamate,asparagine, gamma aminobutyric acid, glycine (and glycine buffer),glutamic acid, glutamate, sodium glutamate, histidine (and histidinebuffer), lysine glutamate, lysine aspartate, arginine aspartate,imidazole, serine, threonine, alanine, polyglutamic acid, polylysine,glycylglycine and the like, including hydroxypropyl and galactosederivatives. In one particularly preferred embodiment, L-argininemonohydrochloride, L-methionine and L-lysine monohydrochloride are used.

Cryoprotectants/Lyoprotectants

Various cryoprotectants/lyoprotectants can be used in the presentinvention. Suitable cryoprotectants structure water molecules such thatthe freezing point is reduced and/or the rate of cooling necessary toachieve the vitreous phase is reduced. They also raise the glasstransition temperature range of the vitreous state. These include, butare not limited to: dimethylsulfoxide (DMSO), dextran, sucrose,1,2-propanediol, amino acids/salts such as, glycine, lysine, arginine,aspartic acid, histidine, proline, etc., glycerol, sorbitol, sodiumchloride, fructose, trehalose, raffinose, stachychose, propylene glycol,2,3-butanediol, hydroxyethyl starch, polyvinylpyrrolidone (PVP), PEG'sand similar compounds, protein stabilizers, such as human serum albumin,bovine serum albumin, bovine gamma globulin, gelatin (or derivatives,such as Prionex, etc.), dextrose, glucose, maltose, arabinose, lactose,inositol, polyols (such as sorbitol, xylitol, erithritol, glycerol,ethylene glycol, etc.), tetramethylglucose, sodium sulfate,cyclodextrins and combinations thereof. Lysine and arginine arepreferred cryoprotectants/lyoprotectants.

Surfactants/Solubilizers/Surface Active Agents

Peptides are susceptible to physical degradation through denaturation,aggregation, precipitation, container surface adsorption and/oragitation induced denaturation. The addition of a nonionic surfactant,such as polysorbate, to the formulation, may reduce the interfacialtension or aid in solubilization thus preventing or reducingdenaturation and/or degradation at air/liquid or liquid/solid interfacesof the product in solution.

Surfactants/solubilizers include compounds such as free fatty acids,esters of fatty acids with polyoxyalkylene compounds likepolyoxypropylene glycol and polyoxyethylene glycol; ethers of fattyalcohols with polyoxyalkylene glycols; esters of fatty acids withpolyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate;glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyalkylated; mono-, di- and triglycerides of saturated orunsaturated fatty acids; glycerides or soya-oil and sucrose; sodiumcaprolate, ammonium sulfate, sodium dodecyl sulfate (SDS), Triton-100and anionic surfactants containing alkyl, aryl or heterocyclicstructures.

Examples of preferred surfactants/solubilizers for use in the presentinvention include, but are not limited to, pluronics (e.g., Lutrol F68,Lutrol F127), Poloxamers, SDS, Triton-100, polysorbates such as TWEEN®20 and TWEEN® 80, propylene glycol, PEG and similar compounds, Brij58(polyoxyethylene 20 cetyl ether), cremophor EL, cetyl trimethylammoniumbromide (CTAB), dimethylacetamide (DMA), NP-40 (Nonidet P-40), andN-methyl-2-pyrrolidone (Pharmasolve), glycine and other aminoacids/amino acid salts and anionic surfactants containing alkyl, aryl orheterocyclic structures, and cyclodextrins. TWEEN® 20 is the mostpreferred surfactant in formulations of the invention.

Bulking Agents/Tonicity Adjusters

Due to the small amount of sincalide present in the formulations of theinvention, bulking agents/tonicity adjusters are useful to providestructure and support for the active ingredient, sincalide, as well asto provide tonicity. Bulking agents/tonicity adjusters (also calledlyophilization aids) useful in the preparation of lyophilized productsof the invention are known in the art and include mannitol, lactose,potassium chloride, sodium chloride, maltose, sucrose, PEG's (such as,for example, PEG 300, PEG 400, PEG 3350, PEG 6000, PEG 8000 and thelike, etc.), trehalose, raffinose, dextrose, polygalacturonic acidgalacturonic acid, amino acids (including amino acid salts) such aslysine, arginine, glycine, galactose, etc.), cyclodextrins, such ashydroxypropyl-γ-cyclodextrin (HP-γ-CD), dextran, Ficoll, andpolyvinylpyrrolidone (PVP). Of these, D-mannitol is the most preferredbulking agent/tonicity adjuster for use with the invention.

Other Excipients

Other excipients, which may optionally be used in the formulations ofthe invention include preservatives (e.g., benzalkonium chloride),osmolality adjusters (e.g., dextrose), lyoprotectants (e.g., sodiumsulfate), solubilizers, tonicity adjusters (e.g. sodium chloride), cakeforming agents, complexing agents, and dissolution aids. A listing ofvarious excipients that can be used in sincalide formulations forparenteral administration can be found in, for example, The Handbook ofPharmaceutical Additives, Second Edition, edited by Michael & Irene Ash;Remington's Pharmaceutical Sciences, (18^(th) Edition), edited by A.Gennaro, 1990, Mack Publishing Company, Easton, Pa. and Pollock et al.;Strickly, Robert G., Parenteral Formulations of Small MoleculesTherapeutics Marketed in the United States (1999)—Part I, PDA Journal ofPharmaceutical Science and Technology, 53(6):324 (1999); Strickly,Robert G., Parenteral Formulations of Small Molecules TherapeuticsMarketed in the United States (1999)-Part II, PDA Journal ofPharmaceutical Science and Technology, 54(1):69 (2000); ParenteralFormulations of Small Molecules Therapeutics Marketed in the UnitedStates (1999)-Part III, PDA Journal of Pharmaceutical Science andTechnology, 54(2):154 (2000); Nema, Sandeep, et al., Excipients andTheir Use in Injectable Products, PDA Journal of Pharmaceutical Scienceand Technology, 51(4): 166 (1997); Wang, Y. J., et al., ParenteralFormulations of Proteins and Peptides: Stability and Stabilizers(Technical Report No. 10), Journal of Parenteral Science and Technology,Vol. 42 (2S), Supplement 1988; Carpenter, J. et al., Freezing- andDrying-Induced Perturbations of Protein Structure and Mechanisms ofProtein Protection by Stabilizing Additives, in Drugs and ThePharmaceutical Sciences, Louis Rey and Joan C. May., eds., MarcelDekker, Inc. New York, N.Y. (1999); Michael J. Pikal, Mechanisms ofProtein Stabilization During Freeze-Drying and Storage: The RelativeImportance of Thermodynamic Stabilization and Glassy State RelaxationDynamics, in Drugs and The Pharmaceutical Sciences, Louis Rey and JoanC. May., eds., Marcel Dekker, Inc. New York, N.Y. (1999); Shah, D., etal., The Effects of Various Excipients on the Unfolding of BasicFibroblast Growth Factor, PDA Journal of Pharmaceutical Science &Technology, 52(5):238 (1998); Powell, M. F., et al., Compendium ofExcipients for Parenteral Formulations, PDA Journal of PharmaceuticalScience & Technology, 52(5):238 (1998); and Inactive Ingredient Guide,Div. Of Drug Information Resources, FDA, CDER, January 1996; Handbook ofInjectable Drugs, Edition 8, Am. Soc. Hospital Pharmacists, 1994, L. A.Trissel.

Formulation Kits

Kits of the present invention preferably comprise one or more vialscontaining the sterile formulation of a predetermined amount ofsincalide, a lyophilization aid or bulking agent/tonicity adjuster, oneor more stabilizers, a surfactant, a chelator, and a buffer. The one ormore vials that contain all or part of the formulation can independentlybe in the form of a sterile solution or a lyophilized solid. Bufferingagents useful in the preparation of formulation kits of the inventionare discussed herein and include, for example phosphate, citrate,sulfosalicylate, and acetate, and amino acids (including amino acidsalts). Dibasic potassium phosphate is a preferred buffer in sincalideformulations of the invention. The kits may also include a fluidportion, for example water or saline, for reconstitution of theformulation prior to injection.

Lyophilization aids or bulking agent/tonicity adjusters useful in thepreparation of lyophilized kits include those dicussed above,particularly, mannitol, lactose, sodium chloride, maltose, sucrose,PEG's, galaturonic acid, polygalcturonic acid, cyclodextrins, such ashydroxypropyl-γ-cyclodextrin (HP-γ-CD) and the like, dextran, aminoacids (including amino acid salts), Ficoll, and polyvinylpyrrolidone(PVP). Of these, mannitol, sodium chloride, maltose, sucrose, PEG's,HP-γ-CD, and dextran are preferred bulking agerints/tonicity adjustersfor use with the invention, with mannitol being the most preferred.

As discussed, a component in a formulation kit can also serve more thanone function. For example, an excipient which serves as a stabilizer mayalso serve as the chelator and an excipient which serves as a bulkingagent may also serve as a tonicity adjuster. In addition, in someembodiments, the excipients are all in dry powder form, or all in liquidform while in other embodiments, some of the excipients are in dry formand others are in a fluid portion included in or sold separately fromthe kit.

A particularly preferred kit of the invention contains: about 0.005 mgsincalide, about 170 mg D-mannitol, less than or equal to 0.01 mg TWEEN®20, about 2 mg DTPA, about 0.04 mg sodium metabisulfite, about 9 mgpotassium phosphate (dibasic) about 4 mg L-methionine, about 15 mgL-lysine monohydrochloride, and about 30 mg L-argininemonohydrochloride.

Therapeutic/Diagnostic Uses

Sincalide is a synthetic analog of the endogenously produced hormonecholecystokinin (CCK-8). CCK-8 acts on receptors within the gallbladderwall causing it to contract, cleaning out any remaining sludge or bilethat may have accumulated within the gallbladder. CCK-8 increases bileflow and small and large bowel motility, causes the pyloric sphincter tocontract and increases pancreatic enzyme secretion. CCK-8 also causesdelayed biliary to bowel transit. Sincalide has a more rapid physiologiceffect on the gallbladder in terms of contraction and relaxation thanthe endogenous hormone (CCK-8) produced by the body, making sincalideformulations useful as diagnostic aids for hepatobiliary imaging, whenadministered alone or in conjunction with a hepatobiliary imaging agent.For example, sincalide may be administered before and/or afterdiagnostic imaging (such as, for example, magnetic resonance imaging,scintigraphic imaging, ultrasound imaging, etc.) to improvevisualization and/or diagnosis of various disease states.

In one embodiment, hepatobiliary imaging can be performed using, forexample, hepatobiliary scintigraphy, an instrumental imaging tool usedin the diagnosis and evaluation of hepatobiliary disease. Detection ofdiseases, such as acute and chronic cholecystitis, biliary obstruction,bile leaks, and other forms of hepatobiliary disease, help the physicianto better determine the appropriate course of treatment and managementof the patient suffering from a suspected hepatobiliary pathology.

As explained below, the indications for use of sincalide in conjunctionwith hepatobiliary imaging include (a) pretreatment of patients who havenot eaten for more than 20 to 24 hours prior to imaging (in order toempty the gallbadder (GB) of non-radiolabelled bile) and (b) use in theanalysis of gallbladder motor function, including the determination ofGBEF (gallbladder ejection fraction).

It is important to properly prepare the patient prior to hepatobiliaryimaging in order to achieve high quality imaging and reduce the numberof false positive and negative results. Preferably, patients should havenothing to eat for 4 to 12 hours prior to hepatobiliary imaging.Prolonged fasting, however, may result in false positive test results(i.e. failure to visualize the gallbladder). If a patient has not eatenfor more than 24 hours, the patient is preferably pretreated withsincalide by administration of the sincalide formulation describedherein prior to imaging. Typically, the gallbladder contracts within 15minutes after sincalide injection and the hepatobiliary imaging agent(e.g., radiotracer) is injected 30 minutes later. The gallbladder isthen emptied and is better able to take up and accumulate imaging agent(e.g., radiotracer), which helps to reduce the number of false positivestudies.

The preferred radiopharmaceuticals used for hepatobiliary imaginginclude, but are not limited to, Tc 99m IDA (Iminodiacetic acid)analogs, such as Tc-99m mebrofenin (CHOLETEC®), Tc-99m disofenin(DISIDA), and Tc-99m lidofenin (see also U.S. Pat. No. 4,418,208).Tc-99m mebrofenin is a preferred hepatobiliary imaging agent. Methodsfor coadministration of Tc 99m IDA (Iminodiacetic acid) analogs with CCKand sincalide are known in the art and described in, for example,Ziessman H A., Cholecystokinin cholescintigraphy: victim of its ownsuccess? J. Nucl. Med. 1999, 40:2038-2042; Krishnamurthy S., et al.,Gallbladder ejection fraction: A decade of progress and future promise.J. Nucl. Med. 1992, 32:542-544; Krishnamurthy G T., et al., Quantitativebiliary dynamics: introduction of a new noninvasive scintigraphictechnique. J. Nucl. Med. 1983; 24:217-223; Mesgarzadeh M., et al.,Filling, post-cholecystokinin emptying and refilling of normalgallbladder: effects of two different doses of CCK on refilling: ConciseComm. J. Nucl. Med. 1983, 24:666-671; Krishnamurthy G T., et al., Thegallbladder emptying response to sequential exogenous cholecystokinin,Nucl. Med. Com., 1984, 5 (1) pp 27-33; Krishnamurthy G T., et al.,Detection, localization, and quantitation of degree of common bile ductobstruction by scintigraphy, J. Nucl. Med. 1985, 26:726-735; Fink-BennetD., et al., Cholecystokinin cholescintigraphic findings in the cysticduct syndrome, J. Nucl. Med. 1985, 26:1123-1128; Fink-Bennet D., Therole of cholecystogogues in the evaluation of biliary tract disorders.Nucl. Med. Ann. 1985, Lenny Freeman and Heidi Weissman, eds., New York,Raven Press, 1985, pp. 107-132; Newman P., et al., A simple techniquefor quantitation cholecystokinin-HIDA scanning. British J. of Radiology,vol. 56, pp. 500-502, 1983; Pickleman J., et al. The role of sincalidecholescintigraphy in the evaluation of patients with acalculousgallbladder disease. Archives of Surgery, vol. 120, 693-697; Ziessman, HA., et al., Calculation of a gallbladder ejection fraction: Advantage ofcontinuous sincalide infusion over the three-minute infusion method. J.Nucl. Med. 1992, 33:537-41; Sitzmann, J V., et al., Cholecystokininprevents parenteral nutrition induced biliary sludge in humans, Surg.Gynecol. Obstet. 170:25-31, 1990; Teitelbaum D H., et al., Treatment ofparenteral nutrition-associated cholestasis withcholecystokinin-octapeptide. J. Pediatr. Surg. 30:1082, 1995.

After administration of the hepatobiliary imaging agent, thehepatobiliary system of the patient can be imaged using an appropriatedetection device. When a Tc-99m IDA (Iminodiacetic acid) analog, such asCHOLETEC® is used as an imaging agent, a gamma camera can be employed toscan the body of the patient for radioactivity. Imaging of thegallbladder allows for the non-invasive measurement and analysis ofvarious biliary motor functions, including the gallbladder ejectionfraction (GBEF). Measurement of GBEF is clinically valuable in thediagnosis and management of certain gallbladder-related disorders,including chronic acalclulous cholecystitis (CAC). In particular, lowGBEF has been found to have a >90% positive predictive value for CAC.Other changes in biliary dynamics may be used in the diagnosis of avariety of biliary disorders.

Methods for determining GBEF scintigraphically are known in the art, andare described in, for example, the references cited above. Sincalideaids in the analysis of biliary function, including the measurement ofGBEF, through its physiological effects on the gallbladder, e.g. itability to induce gallbladder contraction and emptying. One techniquefor measuring GBEF is to administer sincalide slowly as a 1-3 minuteinfusion and to calculate GBEF at the end of about 20 minutes.Alternatively, sincalide may be infused rapidly as a bolus, or as aslower continuous infusion ranging from 15 to 60 minutes. By inducingcertain biliary functions during hepatobiliary imaging, sincalide aidsin the identification of anomalies in such functions, which may beindicative of certain hepatobiliary diseases.

Administration of sincalide formulations can be via IV or IM injections:For IV administration the dose can be administered as a bolus or slowinjection over time optionally with the aid of an infusion pump. Thedose for IV administration is typically 0.005 to 0.04 μg/kg (bolusinjection) or 0.005 μg/kg in a series of 4-three minute injections. Adose of 0.02-0.04 μg/kg IV over 2-3 minutes, but up to 1 hour isdescribed in the art. Injection rates of 0.58 μg/kg/hour can also beemployed with the use of an infusion pump. Other regimens starting at 10ng/kg/hr and increasing to 160 ng/kg/hr are also known in the art. Bolusinjection is not recommended in every case, but injection of 0.02 to0.04 μg/kg over 2-3 minutes even up to 15 min. can be used to avoidspasm of the cystic duct or GB.

Doses for IM administration are generally higher and range from 0.1 to0.4 pig/kg. In one embodiment the 0.4 μg/kg IM dose is generallypreferred resulting in the greatest GB response with the fewest sideeffects. Further details on administration are provided in, for example,Mesgarzadeh M., et al., Filling, post cholecystokinin emptying andrefilling of normal gallbladder: effects of two different doses of CCKon refilling, J. Nucl. Med. 1983, 24:666-671; Ziessmann H A., et al.,Calculation of a gallbladder ejection fraction: Advantage of continuoussincalide infusion over the three-minute infusion method. J. Nucl. 1992,33:537-541; Pickleman J, et al., The role sincalide cholescintigraphy inthe evaluation of patients with acalculous gallbladder disease. Archivesof Surgery, vol. 120, 693-697; Krishnamurthy G T., et al., Thegallbladder emptying response to sequential exogenous cholecystokinin,Nucl. Med. Com., 1984, 5 (1) pp 27-33; Krishnamurthy G T., et al.,Quantitative biliary dymanics: introduction of a new noninvasivescintigraphic technique. J. Nucl. Med. 1983, 24:217-223; Fink-Bennet D.,The role of cholecystogogues in the evaluation of biliary tractdisorders. Nucl. Med. Ann. 1985, Lenny Freeman and Heidi Weissman, eds.,New York, Raven Press, 1985, pp. 107-132; Balon H. R., et al. Society ofNuclear Medicine procedure guideline for hepatobiliary scintigraphy.

The sincalide formulations of the invention are also useful for treatingpatients receiving total parenteral nutrition (TPN). TPN induces biliarysludge, the development of cholestasis, and the formation of gall stonesand other gallbladder related complications. Indeed, TPN associatedcholestatis (TPN-AC) can be a fatal in some instances. The clinicalimplications of TPN-AC include increased rates of sepsis, cirrhosis,declined lymphocyte function, obstructive jaundice, liver failure, andincreased mortality. Although the mechanisms by which these disordersdevelop have not been definitely established, biliary stasis, thereduction in gallbladder emptying, bile flow, and bile acid secretionthat accompanies TPN, has been implicated in the pathogenesis of TPN-ACand other TPN-associated complications. By promoting biliary contractionand emptying, the administration of sincalide to a TPN patient can helpto treat and prevent diseases and other complications associated withprolonged TPN.

For TPN patients the dose of 0.05 μg/kg is typically given IV over 10minutes as a daily infusion. In infants, to treat high bilirubin levelsthe dose is 0.02 μg/kg IV or IM twice or 3 times daily with dosesincreasing up to 0.32 μg/kg. CCK induces not only GB contraction butalso increases intrahepatic bile flow. Information on the treatment ofTPN-patients is provided in, for example, Sitzmann, J V., et al.,Cholecystokinin prevents parenteral nutrition induced biliary sludge inhumans, Surg. Gynecol. Obstet. Vol. 170:25-31, 1990; Moss R L., et al.,New approaches to understanding the etiology and treatment of totalparenteral nutrition-associated cholestasis, Surg. Gynecol. Obstet. Vol.8:140-147, 1999; Teitelbaum D H., et al., Treatment of parenteralnutrition-associated to cholestasis with cholecystokinin-octapeptide. J.Pediatr. Surg. 30:1082, 1995; Teitelbaum D H. Parenteralnutrition-associated cholestasis, Current Opinion in Pediatrics 1997,9:270-275; Teitelbaum D H., et al., Parenteral nutrition-associatedcholestasis. Seminars in Pediatric Surgery, Vol. 10, pp. 72-80.

The present invention is illustrated by the following examples, whichare in no way intended to be limiting of the invention.

Example 1 Effect of Buffering Agent and Formulation pH on SincalideFormulations

Experiments were conducted to determine the effect of pH on the chemicalstability of sincalide. Chemical instability, or degradation, may becaused by, for example, oxidation, reduction, deamidation, hydrolysis,imide formation, racemization, isomerization, and/or β-elimination. Toexamine the effect of pH on sincalide in phosphate buffer solution,solutions of sincalide (≈1.7 μg/mL) were prepared in 35 mM phosphatebuffer and pH-adjusted with either dilute HCl or NaOH for final pHvalues ranging from 3.0-9.1. Using reverse-phase HPLC (RP-HPLC) withgradient elution and UV detection at 215 nm, sincalide stability insolution was assessed by measuring the recovery of sincalide at 0, 6,and 24 hours after pH adjustment.

Results of the 24-hour study on the stability of sincalide in phosphatebuffer over the pH range of 3.0-9.1 are summarized in Table 3 and alsorepresented graphically in FIG. 5. By measure of the percentagerecovery, sincalide was stable in 35 mM phosphate buffer solution at pHvalues ranging from 5.0-9.1 over a 24-hour period. At pH values <5.0,sincalide degradation was evident even at the initial time point.

TABLE 3 Results of pH Study of Sincalide in 35 mM Phosphate BufferAverage % Sincalide Recovery pH n 0 Hours 6 Hours 24 Hours 3.0 2  95.2 ±0.4  93.4 ± 0.4 90.8 ± 1.2 4.0 2  93.0 ± 0.6  92.6 ± 1.6 85.5 ± 3.0 5.04 100.0 ± 2.7  99.8 ± 1.3 97.3 ± 1.8 5.5 2 100.7 ± 0.0 102.1 ± 0.3 101.6± 0.6  6.0 2  97.8 ± 0.4  99.8 ± 0.2 99.8 ± 1.0 6.5 2  98.8 ± 0.4 100.7± 0.3 99.6 ± 0.1 7.0 2 101.0 ± 0.0 101.0 ± 1.8 100.2 ± 1.2  7.5 2 101.0± 0.2 101.2 ± 0.8 100.4 ± 0.0  9.1 5 101.3 ± 2.3 101.1 ± 1.6 99.7 ± 0.9

Based on the results shown in Table 3, phosphate was selected as thebuffering agent of choice due to a lack of interaction with sincalideand an ideal buffering capacity in the physiological pH range.Subsequently, experiments using phosphate in the formulation shown inTable 4 over the stable pH range established above were performed.Briefly, solutions of sincalide containing the following components (inthe concentrations indicated in Table 4) were prepared: sincalide,D-mannitol, L-arginine, L-methionine, L-lysine, sodium metabisulfite,polysorbate 20, pentetic acid and dibasic potassium phosphate.

TABLE 4 Components of a Sincalide Formulation for Example 1Concentration Component (mg/vial) Function Sincalide 0.0050 ActiveD-Mannitol 170.0 Bulking Agent/Tonicity Adjuster L-ArginineMonohydrochloride 30.0 Stabilizer L-Methionine 4.0 Stabilizer L-LysineMonohydrochloride 15.0 Stabilizer Sodium Metabisulfite 0.040 StabilizerPolysorbate 20 (TWEEN ®-20) <0.01 Surfactant Pentetic Acid (DTPA) 2.0Chelator Dibasic Potassium Phosphate 9.0 Buffer

Solutions were pH-adjusted from 5.5-8.5 with dilute HCl or NaOH, andwere evaluated for stability by measuring the sincalide recoveries at 0,4, and 8 hours after pH adjustment, using RP-HPLC with gradient elutionand UV detection at 215 nm, as described above. The results of an 8-hourstudy on the stability of sincalide in the above formulation over the pHrange of 5.5-8.5 are summarized in Table 5 and also representedgraphically in FIG. 6.

TABLE 5 Results of pH Study of a Preferred Lyophilized SincalideFormulation of the Invention Average % Sincalide Recovery pH n 0 Hours 4Hours 8 Hours 5.5 2 99.7 ± 0.2 98.5 ± 0.1 98.1 ± 0.0 6.0 2 97.4 ± 0.598.0 ± 0.1 98.0 ± 0.2 7.0 2 98.4 ± 0.1 98.1 ± 0.1 97.5 ± 1.3 8.0 2 97.2± 0.6 95.4 ± 0.4 96.4 ± 0.2 8.5 1, 2, 2 99.2 98.0 ± 0.0 99.5 ± 0.9

No distinct pH-dependent related trends in initial sincalide recoverywere observed over the pH range studied. Any fluctuation in sincaliderecovery over time can be attributed to normal assay variability and notdegradation. Sincalide stability in this formulation is furthersupported by analyses of the chromatographic profiles for the presenceof sincalide-related degradants which were consistent at 1.2-1.6%(impurity index) over the 8-hour study from pH 5.5-8.5. A bulk batchsolution of sincalide formulation was prepared containing 25 mMphosphate, as a buffering agent, at a target pH value of 6.8 (range6.7-6.9). Reconstitution of the lyophile with 5 mL of water isequivalent to 10 mM phosphate in the drug product. The data demonstratesolution stability over a physiologically compatible pH range andsupport a preferred pH of 6.0-8.0 for reconstituted sincalide.

Example 2 Effect of Chelators on Sincalide Formulations

As shown in FIG. 1, the amino acid composition of sincalide includes twomethionine (Met) residues which are designated as Met 3 and Met 6 in thestructural sequence. Experiments were performed to determine whetherthese residues, as present in sincalide, were susceptible to oxidationby free metals. These experiments also examined the role of DTPA as aformulation excipient to chelate metals and thereby inhibit sincalideoxidation. FIGS. 2-4 show the three oxidized forms of sincalidecontaining either mono- or disulfoxides. As shown in Table 6,experimental formulations (without amino acids) at pH 6.5-7.0, with 1 mMDTPA (0.39 mg DTPA/mL) and without DTPA were prepared to evaluatepotential oxidative effects due to the presence of metals.

TABLE 6 Sincalide Formulations Used in Example 2 (without Amino Acids)Bulk Concentration Concentration Component (mg/vial) (mg/mL) Sincalide0.0050 0.0025 D-Mannitol 170.0 85.0 L-Arginine Monohydrochloride 0 0L-Methionine 0 0 L-Lysine Monohydrochloride 0 0 Sodium Metabisulfite0.040 0.02 Polysorbate 20 <0.01 2.5 × 10⁻⁶ Pentetic Acid (DTPA) (+)/(−)1.0/0 Dibasic Potassium Phosphate 9.0 4.5

The experimental formulation (25 mL) solution with (+) and without (−)DTPA were individually spiked with nine metal ions, as summarized inTable 7.

TABLE 7 Evaluation of Metal Ions for Oxidative Effects on SincalideVolume (μL) of 1 mM DTPA Metal Ion Metal Ion (+) with/ Metal StandardConcentration (−) without Aluminum 100 1.48 mM + (Al³⁺)   40 ppm −Chromium 25 0.19 mM + (Cr³⁺)   10 ppm − Copper 100 0.63 mM + (Cu²⁺)   40ppm − Iron 25 0.18 mM + (Fe³⁺)   10 ppm − Lead 100 0.19 mM + (Pb²⁺)   40ppm − Magnesium 50 0.82 mM + (Mg²⁺)   20 ppm − Manganese 25 0.18 mM +(Mn²⁺)   10 ppm − Nickel 100 0.68 mM + (Ni²⁺)   40 ppm − Zinc 100 0.61mM + (Zn²⁺)   40 ppm −

The metal-containing solutions were analyzed within 8 hours forsincalide and related oxidized forms by RP-HPLC with gradient elutionand UV detection at 215 nm, as described above. FIG. 7 shows the effectsof the nine metals in the presence and absence of DTPA on the formationof sulfoxides (Met 3 and Met 6). These data show that, with theexception of Cr³⁺, the amounts of sincalide Met 3 and Met 6monosulfoxides increase in the presence of certain metals and in theabsence of DTPA, while the presence of DPTA has an inhibitory effect onthe formation of sincalide Met 3 and Met 6 monosulfoxides. Copper andmanganese, in the absence of DTPA, have the greatest oxidative effect onthe methionine residues of sincalide resulting in combined weightpercentages of Met.3 and Met 6 monosulfoxides (vs sincalide) of 85.5 and128.9, respectively. In addition to the presence of sincalide Met 3 andMet 6 monosulfoxides (t_(R) 14.8 min. {doublet} and t_(R)≈18 min.),formation of sincalide disulfoxide (t_(R)≈6.5 min.) was also noted inthe cases of copper and manganese, but not with the other metals.

Chromatograms of formulations spiked with copper or manganese (FIGS.8-11) and with or without DTPA also support this conclusion. Theanalyses of the chromatographic profiles indicate that levels of DTPA at1 mM (0.39 mg DTPA/mL) protect sincalide from metal-catalyzed oxidationto sulfoxides. As trace metals often arise in formulations as a resultof excipient impurities and/or stopper extractables, the results of thestudy support the use of pentetic acid (DTPA) as a formulation excipientto chelate trace levels of free metals, thus reducing the formation ofsincalide methionine mono- and disulfoxides and inhibiting thedegradation of sincalide in solution. Sincalide formulations wereprepared containing 2 mg DTPA/vial, equivalent to 1 mM uponreconstitution with 5 mL.

Example 3 Effect of Surfactants on Sincalide Formulations

During the preliminary developmental studies of a new formulation thatconsisted of bulking agent/tonicity adjuster, buffer, salt, chelator,and sincalide, it was observed by HPLC analysis that the recovery of theactive pharmaceutical ingredient, sincalide, in the bulk solution wassensitive to standing open to air. For example, when usingreversed-phase gradient elution HPLC with UV detection at 215 nm tomonitor sincalide potency, a substantial decrease of 50-60% in sincaliderecovery was observed in unstoppered vials with a 2-mL fill of bulksolution either stirred or left standing open to air for 17 hours.Although to some extent, this sincalide decrease can be accounted for byan increase in the presence of sincalide mono- and disulfoxidedegradants, these represent only a very minor percentage of thedecreases noted. Thus the decrease in recovery is thought to beattributed to either adsorption/denaturing or air/liquid interfaceeffects. To minimize sincalide degradation associated with surfaceadsorption, surfactants are added as formulation excipients in bulk andlyophilized formulations of sincalide.

Sincalide formulations consisting of a bulking agent/tonicity adjuster(D-mannitol), buffer (mono- and dibasic potassium phosphate), salt(sodium/potassium chloride) for tonicity, chelator (pentetic acid), andactive ingredient (sincalide) were prepared using varying concentrationsof the nonionic surfactant, polysorbate 80 (TWEEN® 80). Bulk solutionand reconstituted lyophilized samples were either stoppered immediatelyor left unstoppered for 17 hours, and were assayed for sincaliderecovery by reversed-phase gradient elution HPLC at 2.15 nm.

As shown in Table 8, the effect of TWEEN® 80 is more apparent informulations that have been exposed to air. For bulk and reconstitutedlyophilized formulations, the data show decreases in sincalide recoveryof 50% and =20%, respectively, when compared to correspondingformulations containing a TWEEN® 80 concentration of 1 mg/mL. Lowsincalide recoveries in closed bulk and reconstituted lyophilizedformulations without TWEEN® 80 are also evident, but not nearly assubstantial (4-8%) as the exposed formulations. These preliminaryscreening studies on the influence of TWEEN® 80 concentration indicatethat <1 mg/mL bulk may be optimal.

TABLE 8 Sincalide Recovery in Formulations With and Without TWEEN ® 80Formulation Description Test TWEEN ® 80 Sincalide % (mg/mL Bulk)Condition Conc. (mg/mL) Recovery D-Mannitol (75.0), Bulk; open 1.0 97.0KH₂PO₄ (3.25), (~17 h) 0.0 47.0 K₂HPO₄ (1.0), Bulk; 1.0 100.0 NaCl(5.0), closed 0.0 96.0 DTPA (1.0), Lyophilized; 1.0 91.3 Sincalide(0.0025), open (~17 h) 0.1 98.2 TWEEN ® 80 (0; 0.1; 0.01 98.3 1.0) 0.078.4 Lyophilized; 1.0 90.2 closed 0.1 98.1 0.01 97.8 0.0 92.3

To compare the effects of two nonionic surfactants, sincalideformulations (75 mg/mL D-mannitol, 6.0 mg/mL KCl, 3.25 mg/mL KH₂PO₄, 1.0mg/mL K₂HPO₄, 1.0 mg/mL DTPA, 0.0025 mg/mL sincalide (Bulk formulation))were prepared using either TWEEN® 20® or TWEEN® 80 in varying amounts.The results of this experiment are presented in Table 9.

TABLE 9 Effect of Surfactants on Sincalide Recovery TWEEN ®Concentration Sincalide Sincalide Formulation (μg/mL Bulk) Recovery (%)TWEEN ® 80 A 7.5 95.4 B 5.0 96.3 C 2.5 98.6 G 0 94.1 TWEEN ® 20 D 7.599.5 E 5.0 101.3 F 2.5 98.7 G 0 94.1

As shown in Table 9, the data indicate that the presence of trace levels(2.5-7.5 μg/mL) of either TWEEN® 80 or TWEEN® 20 has a beneficial effecton the recovery of sincalide, when compared to formulations withoutsurfactant. However, the sincalide recoveries (98-102%) withformulations containing TWEEN® 20 are consistently higher thanrecoveries (95-98%) with TWEEN® 80, and thus TWEEN® 20 is a preferredsurfactant.

An additional experiment was performed to confirm the effect of theconcentration of TWEEN® 20 in terms of sincalide recovery in both airexposed and sealed bulk formulation. Sincalide recovery, determined forbulk formulation (75.0 mg/mL D-mannitol, 6.0 mg/mL KCl, 3.25 mg/mLKH₂PO₄, 1.0 mg/mL DTPA, 0.0025 mg/mL sincalide) containing varying tracelevels of TWEEN® 20 stored in open or closed vials using reversed-phasegradient elution HPLC, is shown in Table 10.

TABLE 10 Effect of TWEEN ® 20 Concentration on Recovery of Sincalide inBulk Formulations Sincalide TWEEN ® 20 Concentration Sincalide %Recovery Formulation (μg/mL Bulk) Open Vial Closed Vial D 7.5 100.7100.8 E 5.0 100.0 100.4 F 2.5 99.0 98.2 G 0 89.8 96.1

As shown in Table 10, the bulk formulations containing TWEEN® 20 haveimproved sincalide recoveries over formulations with no TWEEN® 20 andthe sincalide recoveries are independent of the TWEEN® 20 concentrationrange (2.5-7.5 μg/mL bulk) studied. In addition, the air sensitivityrelative to sincalide recovery was eliminated, as both open and closedformulations containing TWEEN® 20 have equivalent sincalide recoveries.Although these data support the use of TWEEN® 20, it was noted that 2-mLfilled vials containing a TWEEN® 20 concentration of 5 μg/mL show slightfoaming in the reconstituted product upon vigorous stirring. To reducefoaming, a lower TWEEN® 20 concentration was evaluated.

As summarized in Table 11, an experiment was conducted on thelyophilized product comparing the recovery of the sincalide in theformulations with TWEEN® 20 (2.5 ng/mL) and without TWEEN®20. In thisExample and the subsequent Examples, mannitol refers to D-mannitol,methionine refers to L-methionine, arginine refers to L-argininemonohydrochloride, and lysine refers to L-lysine monohydrochloride.

TABLE 11 Sincalide Recovery in Reconstituted Formulations With andWithout TWEEN ® 20 Formulation Sincalide % Recovery Description TWEEN ®20 Concentration (mg/mL Bulk) 0 ng/mL 2.5 ng/mL Mannitol (85.0), 94.8 (n= 5) KH₂PO₄ (4.5), 100.0 (n = 2) DTPA (1.0), 100.0 (n = 2) Methionine(2.0),  99.0 (n = 2) Lysine (7.5), Arginine (15.0), Sodium metabisulfite(0.02), Sincalide (0.0025) Average 94.8 99.7 Variance 0.862 0.667 P (T<= t) two-tail 1.6 × 10⁻⁵

Reducing the amount of TWEEN® 20 to a minimal trace concentration (2.5ng/mL) still produced a significant effect on the air/liquid interfaceand eliminated the foaming in the formulation. A statistical two-tailt-test performed on the results showed a significant difference (P<0.05)between 2.5 ng/mL and no TWEEN® 20 in the formulation. Based on thesedata, the effectiveness of TWEEN® 20, polyoxyethylene sorbitanmonolaurate, as a surfactant was established by enhancing the sincaliderecovery and thus maintaining sincalide potency in the formulation. Apreferred formulation of sincalide includes the nonionic surfactantTWEEN® 20 as a trace excipient at a concentration of 2.5 ng/mL of bulkformulation equivalent to 1 ng/mL in the final product whenreconstituted to 5 mL.

Example 4 Effect of Antioxidants on Sincalide Formulations

An experiment was performed to evaluate the addition of antioxidants asstabilizing agents to prevent sincalide oxidation in formulations ofsincalide (formulations for Example 4 contained 85 mg/mL mannitol, 0.005mg/mL TWEEN® 20, 2.75 mg/mL KH₂PO₄, 1.0 mg/mL DTPA, 2.0 mg/mLmethionine, 7.5 mg/mL lysine, 15 mg/mL arginine, 0.0025 mg/mL sincalide(Bulk formulation), except placebos which contained no sincalide.) Theformation of sincalide methionine (Met 3 or Met 6) monosulfoxides,desulfated sincalide and unknown degradants was investigated. Theeffectiveness of sodium metabisulfite, ascorbic acid, cysteine,glutathione, sodium sulfate, benzalkonium chloride, and benzethoniumchloride in inhibiting the degradation of sincalide in terms of theireffect on sincalide recovery and sincalide-related impurities, wasevaluated by HPLC.

The effect of various antioxidants on the stabilization of sincalide wasevaluated on open and sealed sincalide formulations over 15 hours. Theantioxidants were separately added at a concentration of 10 μg/mL towater-reconstituted lyophilized sincalide formulations containing allformulation ingredients except antioxidant. Spiked and unspikedsolutions were pooled, subdivided, and either exposed to or protectedfrom air over 15 hours. The sincalide and total sincalide-relatedimpurities were monitored by reversed-phase HPLC with gradient elutionand UV detection at 215 nm to compare the effectiveness of theantioxidants.

As shown in Table 12, the data at these concentrations indicate thatbenzalkonium chloride and benzethonium chloride had a significantdestabilizing effect on sincalide, while ascorbic acid, cysteine,glutathione, and sodium sulfate were essentially equivalent to thecontrol formulation (no antioxidant). Of all the sincalideformulation/antioxidant combinations examined, the formulation with 10μg sodium metabisulfite/mL showed the highest sincalide potency (98.3%)over 8 hours, and the lowest total sincalide-related impurities (1.79%)through 15 hours. Therefore, sodium metabisulfite is a preferredantioxidant for formulations of the invention.

TABLE 12 Effect of Various Antioxidants (10 μg/mL) on SincalideFormulation Stability % Sincalide % Total Sincalide-Related ImpuritiesAntioxidant Sealed Open Sealed Open (10 μg/mL) 0 h 7 h 14 h 1 h 8 h 15 h0 h 7 h 14 h 1 h 8 h 15 h Control (None) 98.1 98.1 98.1 98.1 98.2 98.21.94 1.95 1.86 1.90 1.85 1.81 Sodium Metabisulfite 98.3 98.3 98.3 98.298.3 98.2 1.67 1.66 1.73 1.76 1.69 1.79 Ascorbic Acid 98.1 98.0 97.898.0 98.0 97.8 1.95 2.05 2.25 2.00 2.01 2.16 Cysteine 98.2 98.1 98.197.8 97.7 98.0 1.85 1.87 1.91 2.20 2.32 2.05 Glutathione 98.1 98.3 98.298.1 98.2 97.9 1.90 1.74 1.82 1.94 1.85 2.13 Sodium Sulfate 98.2 98.198.2 98.3 98.2 98.1 1.76 1.90 1.81 1.70 1.78 1.92 Benzalkonium Chloride97.8 97.7 97.4 82.7 88.4 82.9 2.21 2.34 2.58 17.3 11.6 17.1 BenzethoniumChloride 97.9 98.0 98.0 92.1 88.0 92.6 2.13 1.98 1.96 7.93 12.0 7.36

To optimize the level of sodium metabisulfite in the formulation,lyophilized sincalide formulations were prepared containing four levelsof sodium metabisulfite (0, 10, 30, and 60 μg/vial), as summarized inTable 13. Samples at each concentration were maintained under unstressedand stressed (65° C., 64 hours) storage conditions, and weresubsequently assayed by HPLC. The “% sincalide” was determined, and the“% (Met 6) monosulfoxide” (t_(R)≈19.7 min.) was monitored as anindication of sincalide oxidation. The data are presented in Table 14.

TABLE 13 Sincalide Lyophilized Formulations Formulation No. FormulationDescription 1 complete formulation, no sodium metabisulfite 2 completeformulation, 10 μg sodium metabisulfite/vial 3 complete formulation, 30μg sodium metabisulfite/vial 4 complete formulation, 60 μg sodiummetabisulfite/vial 5 placebo(no sincalide), 40 μg sodiummetabisulfite/vial 6 complete formulation, no sodium metabisulfite 7complete formulation, 40 μg sodium metabisulfite/vial

TABLE 14 Effect of Sodium Metabisulfite Concentration on SincalideOxidation Sodium % (Met 6) Metabisulfite Monosulfoxide % SincalideConcentration Stressed Stressed Formulation (μg/vial) Unstressed (65°C., 64 h) Unstressed (65° C., 64 h) 1 0 0.08 0.20 95.7 95.1 2 10 0.070.09 95.1 96.0 3 30 0.07 0.10 95.0 96.9 4 60 0.06 0.08 95.8 96.2

The addition of sodium metabisulfite up to 60 μg/vial improved sincaliderecovery and inhibited the oxidation of sincalide to the (Met 6)monosulfoxide derivative under stressed conditions. Based on this data,as there was no apparent concentration relationship, 40 μg/vial sodiummetabisulfite was selected as the preferred concentration for the finalformulation, using 30 μg/vial and 60 μg/vial as lower and upper limits,respectively.

Another experiment was conducted under longer-term accelerated storageconditions utilizing a sincalide formulation with the optimizedconcentration (40 μg/vial) of sodium metabisulfite to confirm theprotective effect on the degradation of sincalide. Sincalide lyophilizedformulations with and without the antioxidant from the same batch wereheat-stressed at 40° C. and 60° C. for 6 weeks. Also, formulationswithout sincalide from the same batch were heat-stressed at 40° C. for 8months. The results of the HPLC analyses for % sincalide and % totalimpurity are presented in Table 15.

TABLE 15 Effect of Sodium Metabisulfite on Heat Stress-RelatedImpurities Storage % Sodium Metabisulfite Temp. % Total FormulationConcentration (μg/vial) (6 weeks) Sincalide Impurity 7 40 40° C. 96.73.30 6 0 40° C. 93.4 6.56 7 40 60° C. 89.5 10.51 6 0 60° C. 84.0 16.00

The results of this longer-term accelerated storage experiment furtheremphasized the need for the presence of the excipient sodiummetabisulfite. Sincalide formulations with sodium metabisulfite (40μg/vial) protected against sincalide heat stress-related degradantformation (3.30%), as compared without the antioxidant, which exhibitedseveral elevated sincalide heat stress-related impurities (6.56%). Theseimpurities were confirmed to be sincalide heat stress-related (t_(R)=35to 44 min.), as they were not present in chromatograms of formulationswithout sincalide. Sodium metabisulfite was chosen as a preferredantioxidant and stabilizing agent over ascorbic acid, cysteine,glutathione, sodium sulfate, benzalkonium chloride, and benzethoniumchloride because it provided superior protection in inhibiting theoxidative and heat stress-related degradation of sincalide. A preferredconcentration in the lyophilized formulation is 40 μg sodiummetabisulfite/vial or 8 μg/mL in the reconstituted product.

Example 5 Selection of Bulking Agent/Tonicity Adjuster

Due to the minute amount of the active pharmaceutical ingredient (API),sincalide (5 μg/vial), in the formulations of the invention, the use ofa bulking agent was considered extremely beneficial for providingtonicity as well as for providing both structure and support for theAPI. Experiments were conducted for the selection and optimization ofbulking agent in the sincalide formulations of the invention. Criteriafor evaluation were: an efficient lyophilization cycle, apharmaceutically elegant finished product, enhanced product solubilityand usefulness as an excipient for isotonicity in the reconstitutedproduct. Various concentrations of lactose, lactose/sodium chloride andmannitol were considered, and experimental batches containing theseexcipients were evaluated in terms of cake appearance, osmolality,dissolution rate, and thermal analysis including freeze dry microscopy,and electrical resistance vs. temperature measurements.

Experimental Formulations

Batch A: ingredients: lactose 375 mg/vial, dibasic sodium phosphate 12.0mg/vial, DTPA 2.0 mg/vial, monobasic sodium phosphatel 19.5 mg/vial, and0.005 mg/vial sincalide.

Batch C₁₋₃: ingredients: mannitol 170 mg/vial, dibasic potassiumphosphate 9.0 mg/vial, TWEEN® 20<0.01 mg/vial, methionine 4.0 mg/vial,lysine 15.0 mg/vial, arginine 30.0 mg/vial, sodium metabisulfite 0.04mg/vial, sincalide 0.005 mg/vial, and DTPA 2.0 mg/vial.

Batch D₁: ingredients: lactose 150 mg/vial, dibasic potassium phosphate9.1 mg/vial, DTPA 2.0 mg/vial, monobasic sodium phosphate 9.8 mg/vial,and NaCl 21.0 mg/vial.

Batch E₁: ingredients: lactose 200 mg/vial, dibasic sodium phosphate 7.5mg/vial, DTPA® 2.0 mg/vial and NaCl 17 mg/vial.

Batch F₁₋₂: F₁: ingredients: mannitol 250 mg/vial, dibasic sodiumphosphate 7.5 mg/vial, DTPA 2.0 mg/vial and sincalide 0 mg/vial; and

-   -   F₂: ingredients: mannitol 206 mg/vial, dibasic sodium phosphate        7.5 mg/vial, DTPA 2.0 mg/vial and sincalide 0.005 mg/vial.

Batch H₁₋₂: H₁: ingredients: mannitol 180 mg/vial, dibasic sodiumphosphate 6.0 mg/vial, sincalide 0 mg/vial, NaCl 5 mg/vial and DTPA 2.0mg/vial; and

-   -   H₂: ingredients: mannitol 150 mg/vial, dibasic potassium        phosphate 4.5 mg/vial, sincalide 0.005 mg/vial, NaCl 10 mg/vial        and DTPA 2.0 mg/vial.

Batch I₁₋₂: I₁: ingredients: mannitol 140 mg/vial, dibasic potassiumphosphate 5.5 mg/vial, TWEEN® 20 0.01 mg/vial, methionine 4.0 mg/vial,lysine 60.0 mg/vial, sincalide 0.005 mg/vial and DTPA 2.0 mg/vial; and

-   -   I₂: ingredients: mannitol 170 mg/vial, dibasic potassium        phosphate 5.5 mg/vial, TWEEN® 20 0.01 mg/vial, methionine 4.0        mg/vial, lysine 30.0 mg/vial, sincalide 0.005 mg/vial and DTPA        2.0 mg/vial.

Batch J: ingredients: mannitol 170 mg/vial, dibasic potassium phosphate8.5 mg/vial, TWEEN® 20 0.01 mg/vial, methionine 4.0 mg/vial, lysine 15.0mg/vial, arginine 30.0 mg/vial, Na metabisulfite 0.04 mg/vial, sincalideand DTPA 2.0 mg/vial.

Methods:

-   1. Appearance: Visual assessment of the freeze-dried plug.-   2. Osmolality: Determined by vapor pressure osmometry.-   3. Dissolution: Dissolution time measured by visual inspection under    an inspection light upon reconstitution with 5 mL of water.-   4. Thermal Analysis:    -   a. Electrical resistance vs. temperature measurements:        Electrical resistance measured using a proprietary resistance        instrument, temperature measured using a 32-gauge type T        thermocouple.    -   b. Freeze drying microscopy: Performed using a freeze dry        microscope an Infinivar microscope and color camera.

In the initial investigations lactose was used as a bulkingagent/tonicity adjuster. The formulation as listed in table 16 is basedon a 3-mL fill volume with a high concentration of lactose to achieveisotonicity in the reconstituted product. The osmolality for thisformulation upon reconstitution with 5 mL of water was ˜300 mOsm/kg.

TABLE 16 Lactose Containing Sincalide Formulation (Batch A)Concentration Raw Materials Function (mg/vial) Lactose Bulking Agent/375 Tonicity Adjuster Dibasic Sodium Phosphate Buffer 12.0 MonobasicSodium Phosphate Buffer 19.5 Pentetic Acid Chelator 2.0 Sincalide Active0.005

This experimental formulation, Batch A, with a lyophilization cycle of130 hours (r 5.4 days) showed evidence of meltback in the lyophilizedcakes and had reconstitution dissolution times of ≧9 minutes. The highnumber of vials with poor cake formation and the long freeze dry cyclerequired were attributed to the high concentration of lactose (125mg/mL) in the bulk formulation relative to its solubility and the highfill volume (3-mL) in a small vial.

Studies were undertaken to reduce cycle time and improve productappearance/solubility by modifying the initial lactose formulation withthe use of an additional excipient, sodium chloride, thereby reducinglactose concentration and the fill volume from 3 to 2-mL.

TABLE 17 Lactose/NaCl Containing Sincalide Formulations (Batches D₁ andE₁₋₂) Concentration Raw Materials Function (mg/vial) Lactose BulkingAgent/ 150-200 Tonicity Adjuster Dibasic Sodium Phosphate Buffer 12.0Monobasic Sodium Phosphate Buffer 19.5 Pentetic Acid Chelator 2.0 SodiumChloride Tonicity Adjuster 17-21 Sincalide Active 0.005

Use of NaCl contributed to the isotonicity of the product withosmolality values in the range of 240 to 270 mOsm/kg, while permitting areduction in the concentration of lactose. Varying the amounts oflactose, sodium chloride and sodium phosphate decreased thelyophilization cycle from 130 hours to 96 hours, but did notconsistently improve the appearance of the freeze-dried cake.

Thermal analysis of two experimental formulations with varyinglactose/sodium chloride ratios (Table 18) confirm that the relativelylong lyophilzation cycles for these formulations were due to low primarydrying temperatures in the range of −38° C. to −42° C., resulting inslow sublimation rates at these temperatures. In addition to longlyophilization cycles, the low primary drying temperatures lead toincreased vial-to-vial variation and an increased risk of poor plugappearance with associated solubility issues.

TABLE 18 Thermal Analysis of Experimental Lactose/NaCl FormulationsLactose/NaCl Freezing Temp. Primary Drying Concentration Range (° C.)Temp. Range (° C.) Batch (mg/vial) High Low High Low D₁ 150/21 −32 −39−39 −42 E₁ 200/17 −15 −35 −36 −38

Mannitol, a common excipient for freeze-dried pharmaceuticals, wasselected next for evaluation as bulking agent because of the highmelting temperature of the mannitol/ice eutectic mixture (about −1.5°C.) and its tendency to crystallize from frozen aqueous solutions.Ideally, this leads to shorter primary and secondary drying times,promoting an efficient freeze-drying cycle and a physically stable,pharmaceutically elegant freeze-dried solid. Several bench-scale batcheswere prepared, replacing lactose with D-mannitol while maintainingisotonicity with a 2 mL fill volume, to evaluate the parameters of cycletime and primary drying temperature and the solubility of the solidcake. The freeze dry cycle parameters along with lyophilized productreconstitution times with a 5 mL reconstitution volume are shown inTable 19.

TABLE 19 Effect of Formulation Bulking Agent/Tonicity Adjuster onLyophilization Cycle Optimization and Reconstitution/Dissolution TimeDisso- Formulation Bulking Freeze Dry lution Description AgentOsmolality Cycle Time Batch (mg/vial) (mg/vial) (mOsm/kg) Parameters(sec) F₁ Na₂HPO₄ Mannitol 280 Total Cycle 12-48 (7.5), (250) 85 hr Pri-(n = 10) DTPA mary drying (2.0) @ −34° C. F₂ Na₂HPO₄ Mannitol 240 TotalCycle 22-71 (7.5), (206) 69 hr Pri- (n = 30) DTPA mary drying (2.0) @−25° C.

Lyo-cycle time was reduced from >130 hours for lactose formulations, to˜69 hours for the mannitol formulation, Batch F₂. The cakes from bothformulations, F₁ and F₂ dissolved in 5 mL of water in approximately thesame time range of <1 minute. Increasing the primary drying temperaturefrom ˜−34° C. to −25° C. Batch F₁ vs. Batch F₂ had the desired effect ofreducing the overall cycle time from 85 to 69 hours.

Additional studies were conducted to optimize the mannitol concentrationand lyo-cycle time for a 2-mL fill volume. These studies were carriedout concurrently with formulation development studies to adjust theosmolality to ˜250 mOsm/Kg after reconstitution and to stabilize thepeptide by addition of other excipients to the formulation (Table 20).

TABLE 20 The Effect of Mannitol Concentration on Appearance, Solubilityand Freeze Dry Cycle of Sincalide Formulations Formulation Freeze DryMoisture Appearance/ Description Bulking Agent Osmolality Cycle ContentDissolution Time Batch (mg/vial) (mg/vial) (mOsm/kg) Parameters (%)(sec) H₁ Na₂HPO₄ (6.0), Mannitol (180) 250 Total Cycle: 36 hr ND Solidcake/ DTPA (2.0), NaCl (5.0) 27 hr primary 22-66 Sincalide (0) @ −8° C.(n = 30) H₂ K₂HPO₄ (4.5), Mannitol (150) 240 Total Cycle: 30 hr 1 Solidcake/ DTPA (2.0), NaCl (10.0) 23 hr primary 11-31 Sincalide (0.005) @−10° C. (n = 30) I₁ TWEEN ® (0.01), Mannitol (140) 250 Total Cycle: 59hr 1 Solid cake/ K₂HPO₄ (5.5), 50 hr primary 21-69 Methionine (4.0), @−22° C. (n = 5) Lysine (60.0), DTPA (2.0), Sincalide (0.005) I₂ TWEEN ®(0.01), Mannitol (170) 250 Total Cycle: 33 hr 1 Solid cake/ K₂HPO₄(5.5), 26 hr primary 8-15 Methionine (4.0), @ −12° C. (n = 5) Lysine(30.0), DTPA (2.0), Sincalide (0.005) ND = Not Determined

These results demonstrate that an increase in primary drying temperaturefrom ˜−25° C. to the −8 to −12° C. range significantly reduced cycletimes from 69 to 30 hours and produced solid dry cakes that reconstitutewithin 1 minute.

Additional optimization studies designed to enhance the long termstability of sincalide resulted in a preferred sincalide formulation of170 mg of D-mannitol/vial with the additional excipients (in mg/vial):TWEEN® 20 (0.01), K₂HPO₄ (8.5), methionine (4.0), lysine (15.0),arginine (30.0), DTPA (2.0), and sodium metabisulfite (0.04). Theosmolality of this optimized formulation was approximately 300 mOsm/kgwhen reconstituted with 5 mL of water. Thermal analysis of thisformulation using freeze-dry microscopy and electrical resistance vs.temperature measurements, indicated an upper limit for product primarydrying temperature of −13° C. to −15° C. to achieve acceptable productquality.

To confirm all findings, three scale-up pilot batches, C₁₋₃, of apreferred sincalide formulation, in a fill volume of 2 mL/vial, wereprepared and freeze dried in full-scale production driers to proveprocess transferability from development equipment to productionequipment. The drying cycle for these batches incorporated a primarydrying temperature of −12° C.+3° C. and an overall cycle time of 53-61hours (Table 21).

TABLE 21 Operating Parameters and Final Product Performance of Scale-upPilot Batches Prepared with Mannitol as a Bulking Agent LyophilizationPrimary Total Moisture Dissolution Temp Cycle Time Osmolality PlugContent Time Batch (° C.) (Hrs) (mOsm/kg) Appearance %) (sec) C₁ −12 58300 Solid cake 1 10 C₂ −12 53 300 Solid cake 1 10 C₃ −12 61 300 Solidcake 1 10

The data from these studies support the selection of mannitol as aparticularly preferred bulking agent, preferably in an amount of about170 mg/vial. Using this concentration, the freeze dry cycle is 53-61hours when filled as a 2-mL fill. The finished product is apharmaceutically elegant, solid white cake, which is reconstitutedwithin one minute using 5 mL of water, resulting in a solution with anosmolality of ˜300 mOsm/Kg.

Example 6 Effect of Amino Acids on Sincalide Formulations

During formulation studies it was observed that both exposure to air andlyophilization were areas of concern for scale-up manufacturing due toreduced potency of sincalide in the formulation. The reduced potency wasa result of surface adsorption/denaturation resulting from exposure ofsincalide to air, and yielding degradants via oxidation. Exposure ofsincalide formulations to thermal stress during lyophilization alsoresulted in degradation and reduced recovery of sincalide.

Experiments were conducted to evaluate several amino acids as potentialstabilizers of sincalide, including the non-polar (hydrophobic)methionine residue, aspartic acid and glutamic acid, the polar glycineand cysteine residues, and the basic lysine and arginine amino acids.

Except as otherwise indicated, the formulations used in this example fortesting the efficacy of various amino acids contained the followingingredients (bulk): 75.0 mg/mL mannitol; 3.25 mg/mL KH₂PO₄; 1.0 mg/mLK₂HPO₄; 1.0 mg/mL pentetic acid (DTPA); 5.0 mg/mL NaCl; and the activepeptide, sincalide (0.0025 mg/mL). Initially, the non-polar amino acidL-methionine was evaluated for the reformulation since methionineresidues can act as endogenous antioxidants, or as scavengers byreacting with hydroxyl free radicals and other reactive oxygen species.Thus, methionine could improve the processing stability of sincalideformulations by providing a protectant or antioxidant effect forsincalide and being preferentially oxidized. Table 22 below summarizesthe results obtained during exposure of experimental formulations to airwhen various amounts of L-methionine were added to a formulationcontaining mannitol, sodium chloride, potassium phosphate, and penteticacid. For these experiments, liquid formulations in open and closedvials were used to simulate processing of the product. For formulationin open vials, the recovery of sincalide was improved approximately 60%and the concentration of sincalide-related impurities decreased as thelevel of methionine was increased from 0.0 to 2.0 mg/mL in the bulkformulation.

TABLE 22 Evaluation of Methionine as a Processing Stabilizer for BulkFormulations - Open vs Closed Vials. L-Methionine Sincalide Recovery (%)Related Impurities (%) (mg/mL Bulk) Open Closed Open Closed 2.0 75.595.7 15.0 0.7 0.50 64.7 94.8 19.3 0.8 0.025 35.7 93.9 35.9 1.0 0.00 13.995.7 52.7 1.3

For comparison to the non-polar amino acid methionine as a potentialprocessing stabilizer, polar amino acids such as glycine and cysteinewere also evaluated. Formulations containing these amino acids wereexposed to air in open vials and compared to product in closed vials.The efficacy of these amino acids, in terms of sincalide recovery andsincalide-related impurities, was compared to the improvementspreviously observed for the liquid formulation in the presence ofmethionine. Table 23 presents the sincalide recoveries for experimentalformulations containing variable concentrations of methionine, cysteineor glycine.

TABLE 23 Comparison of Methionine, Glycine and Cysteine as ProcessingStabilizers for Bulk Formulations - Open vs. Closed Vials Amino AcidSincalide Recovery (%) Related Impurities (%) (mg/mL Bulk) Open ClosedOpen Closed L-Cysteine 2.0 50.0 96.0 31.0 1.0 L-Methionine 2.5 82.4 97.410.5 0.7 L-Methionine 2.0 89.9 97.7 6.4 0.7 None 0 37.0 96.0 35.0 1.6L-Glycine 2.5 31.9 93.2 44.9 1.6 L-Glycine 2.0 22.3 92.5 51.1 1.1

Results demonstrated that addition of either cysteine or glycine to abulk formulation containing mannitol, potassium phosphate, sodiumchloride and pentetic acid did not show a significant effect in eitherreduced levels of sincalide impurities or improved recovery of sincalidewhen formulations were exposed to air in open vials.

Lysine, a basic amino acid, was the next amino acid evaluated for use insincalide formulations of the invention. As shown in Table 24,experimental formulations (70-85 mg/mL mannitol, 0.005 mg/mL TWEEN® 20,2.75 mg/mL KH₂PO₄, 1.0 mg/mL DTPA, 2.0 mg/mL methionine, 0.0025 mg/mLsincalide) were prepared to contain varying concentrations of lysine andevaluated for sincalide recovery.

TABLE 24 Evaluation of Lysine as a Stabilizer in Sincalide ReconstitutedFormulations Sincalide Recovery (%) DL-Lysine 1 Week 3 Weeks 5 Weeks(mg/mL Bulk) 5° C. 40° C. 5° C. 40° C. 5° C. 40° C. 0.0 99.6 84.3 95.551.2 NA 25.4 5.0 98.1 95.4 93.6 98.4 92.0 15.0 97.3 97.0 94.3 99.4 93.230.0 96.6 95.0 95.5 97.2 89.7 NA = Not Applicable

After accelerated storage, lyophilized formulations containing lysineresulted in significantly improved recoveries of sincalide compared to alyophilized control formulation without lysine. Formulations containinglysine resulted in 50% and 75% improvements in sincalide recovery after3 and 5 weeks storage at 40° C., respectively, demonstrating that lysinecontributed to the stability of lyophilized formulations when subjectedto thermal stress.

The improved sincalide recoveries observed in the presence of methionineand lysine suggested that other amino acids might also be suitable asbulk additives in the reformulation. Therefore, formulation studiescontinued with the evaluation of two acidic amino acids, aspartic acidand glutamic acid. Table 25 presents sincalide recoveries forexperimental formulations (85.0 mg/mL mannitol, 0.005 mg/mL TWEEN® 20,2.75 mg/mL KH₂PO₄, 1.0 mg/mL DTPA, 2.0 mg/mL methionine, 0.0025 mg/mLsincalide) containing the following amounts of either lysine, asparticacid or glutamic acid.

TABLE 25 Comparison of Lysine, Aspartic Acid and Glutamic Acid asStabilizers in Sincalide Reconstituted Formulations Formulation ProcessAmino Acid Sincalide Recovery (%) Conditions ID (mg/mL) 0 Days 10 Days30 Days Liquid Bulk A DL-Lysine HCl 15.0 99.9 98.4 NA Stored 5° C. BL-Aspartic Acid 11.0 98.2 96.3 C L-Glutamic Acid 12.0 97.3 96.3Lyophilized A DL-Lysine HCl 15.0 NA 98.2 99.1 Cake B L-Aspartic Acid11.0 94.6 92.8 Stressed C L-Glutamic Acid 12.0 95.5 95.7 40° C. EControl 0.0 81.7 53.8 NA = Not Applicable

The results demonstrated that with increasing storage time at 40° C.,lysine consistently provided better protection than either aspartic acidor glutamic acid. The results obtained for lysine also suggested thatarginine, another basic amino acid, or potentially some combination oflysine and arginine, might further enhance protection duringlyophilization and thermal stress. Experimental formulations (85.0 mg/mLmannitol, 0.005 mg/mL TWEEN® 20, 2.75 mg/mL KH₂PO₄, 1.0 mg/mL DTPA, 2.0mg/mL methionine, 0.0025 mg/mL sincalide) were prepared to containvarying concentrations of lysine, arginine, or a combination of lysineand arginine and evaluated for sincalide recovery (Table 26).

TABLE 26 Evaluation of Lysine and Arginine as Stabilizers in SincalideReconstituted Formulations Sincalide Recovery (%) Amino Acid 64 hrs. 1week (mg/mL Bulk) @ 65° C. @ 40° C. DL-Lysine 15.0 88.4 ND L-Arginine17.5 93.0 DL-Lysine 7.50 99.8 L-Arginine 8.75 DL-Lysine 7.5 93.8 96.4L-Arginine 17.5 DL-Lysine 5.0 91.2 ND L-Arginine 11.7 DL-Lysine 7.5 95.1ND L-Arginine 15.0 N/A (control) 0.0 43.3 ND NA = Not Applicable, ND =Not Determined

Results confirmed that after lyophilization and stressing for 64 hours @65° C., approximately 50-70% improvement in sincalide recovery wasobserved for formulations containing lysine, arginine, or a combinationof the two. Formulations containing both lysine and arginine exhibitedthe highest sincalide recovery values, indicating that the combinationof these two amino acids provided a particularly stabilizing effectunder heat-stressed storage conditions. The mid-point combination of 7.5mg/mL of lysine and 15.0 mg/mL of arginine afforded suitable protectionfor the lyophilized and heat-stressed product, resulting in sincaliderecoveries of >95%.

Methionine, lysine and arginine are preferred over polar amino acidssuch as glycine and cysteine and acidic amino acids such as asparticacid and glutamic acid for use as stabilizers in the sincalideformulations of the invention. Methionine improved the processingstability of the formulation, resulting in improved recovery ofsincalide, and the combination of lysine and arginine contributed to thestability of the product during lyophilization and heat-stressing, alsoresulting in improved recovery of sincalide.

Preferred concentrations in lyophilized formulations of the inventionare: methionine (4.0 mg/vial), lysine (15.0 mg/vial) and arginine (30.0mg/vial).

Example 7 Reconstituted Shelf-Life Studies

A. In-Vial Post-Reconstitution Stability

Experiments were performed to determine the post-reconstitutionstability of sincalide in terms of appearance, solubility, particulatematter, color, pH, sincalide assay, desulfated sincalide assay and othersincalide-related impurities through 8 hours at ambient temperature.Lyophilized vials from three 105-L scale-up pilot batches of sincalideformulations were reconstituted with 5.0 mL of purified water.

Testing was conducted at 0, 2, 4, 6, and 8 hours post-reconstitution forappearance, solubility, particulate matter, color, and pH. Testing wasconducted on duplicate vials at 0, 4, and 8 hours post-reconstitutionfor sincalide assay, desulfated sincalide assay and othersincalide-related impurities using reversed-phase HPLC with gradientelution and UV detection at 215 nm.

The test results for appearance, solubility, particulate matter, color,and pH performed at 0, 2, 4, 6, and 8 hours post-reconstitution for thethree sincalide formulation preparations are shown in Table 27.

TABLE 27 Post Reconstitution Test Results Time Solubility Particulate pHPreparation (hr) Appearance (sec.) Matter Color meter 1 meter 2 A 0Clear 20 Complies Colorless 7.2 7.0 2 Clear 20 Complies Colorless 7.17.0 4 Clear 20 Complies Colorless 7.2 7.0 6 Clear 20 Complies Colorless7.2 7.0 8 Clear 20 Complies Colorless 7.2 7.0 B 0 Clear 20 CompliesColorless 7.2 7.0 2 Clear 20 Complies Colorless 7.1 7.0 4 Clear 20Complies Colorless 7.2 7.0 6 Clear 20 Complies Colorless 7.1 7.0 8 Clear20 Complies Colorless 7.1 7.0 C 0 Clear 20 Complies Colorless 7.1 7.0 2Clear 20 Complies Colorless 7.1 7.0 4 Clear 20 Complies Colorless 7.17.0 6 Clear 20 Complies Colorless 7.1 7.0 8 Clear 20 Complies Colorless7.1 7.0

For the three preparations examined (referred to herein as preparationsA, B, and C), no changes were observed in the parameters tested and allresults were within specifications through the 8-hour testing period (85mg/mL mannitol; 2.5×10⁻⁶ mg/mL TWEEN® 20; 4.5 mg/mL KH₂PO₄; 1.0 mg/mLDTPA, 0.02 mg/mL sodium metabisulfite, 2.0 mg/mL methionine, 7.5 mg/mLlysine, 15.0 mg/mL arginine, 0.0025 mg/mL sincalide (Bulk formulation).The HPLC test results for sincalide assay, desulfated sincalide assayand other sincalide-related impurities performed at 0, 4, and 8 hourspost-reconstitution for the three formulation preparations are shown inTable 28.

TABLE 28 Post Reconstitution HPLC Test Results Desulfated SincalideRelated Time Sincalide Sincalide Impurities Preparation (h) (μg/vial)(w/w % sincalide) (% Impurity Index) A 0 4.99, 4.98 0.32, 0.33 1.41,1.32 4 4.99, 4.97 0.32, 0.36 1.40, 1.35 8 4.97, 4.97 0.35, 0.39 1.40,1.34 B 0 5.04, 5.04 0.28, 0.27 1.29, 1.37 4 5.04, 5.03 0.30, 0.29 1.30,1.39 8 5.03, 5.01 0.31, 0.31 1.44, 1.41 C 0 4.97, 4.94 0.36, 0.36 1.48,1.33 4 4.97, 4.94 0.39, 0.37 1.41, 1.37 8 4.97, 4.92 0.44, 0.44 1.46,1.41

All results were within specifications. The sincalide potency wasunchanged over time. The desulfated sincalide and othersincalide-related impurities show only relatively minor increases whichare insignificant with respect to their individual specifications of 2%and 5%, respectively. The study shows that the initial test values ofreconstituted sincalide formulations are representative of resultsobtained throughout the 8-hour shelf life of reconstituted product. Thedata provided demonstrate the post-reconstitution stability of theformulation and support a post-reconstitution shelf-life of 8 hoursunder ambient conditions.

B. Post-Reconstitution Dilution Study

An experiment was performed to determine the stability of sincalideformulations of the present invention that have been reconstituted anddiluted.

Duplicate vials from three 105-L batches of sincalide formulations ofthe invention were reconstituted with 5 mL water. Vial contents werequantitatively transferred (using Sodium Chloride Injection USP torinse) to 100-mL volumetric flasks and up to 8.4 mL of the formulationswere diluted to volume (100 mL) with Sodium Chloride Injection USP.Sincalide potency, pH and visual appearance were tested 1-hour postpreparation. The results of the testing are presented in Table 29.

TABLE 29 Results for Sincalide Formulations to 100 mL With 0.9% Salineat 1 Hour Post-Reconstitution Sincalide Sample Potency ParticulatePreparation No. (μg/vial) pH Appearance Color Matter A 1 4.8 6.9 ClearColorless Free of particles 2 5.0 6.9 Clear Colorless Free of particlesB 1 5.2 6.9 Clear Colorless Free of particles 2 4.9 6.8 Clear ColorlessFree of particles C 1 4.9 6.9 Clear Colorless Free of particles 2 4.96.8 Clear Colorless Free of particles Mean 5.0 6.9 Std. Dev. 0.1 0.1Confidence Interval 4.8-5.1 6.8-6.9 (p = 0.95 and 5 deg. Freedom) CV (%)2.8 0.8

All sincalide potency, pH and appearance results for diluted samples(reconstituted vial contents further diluted to 100 mL) measured 1-hourpost reconstitution were within the product specifications for thereconstituted product (vial reconstituted with 5 mL water).

Example 8

Sincalide Specific Assay using HPLC

An HPLC method was developed and validated for the determination ofsincalide potency, quantitation of desulfated sincalide impurity anddetermination of a sincalide-related impurity index in KINEVAC®. Themethod is suitable for determining the reconstituted stability ofKINEVAC® when reconstituted as per the product package insert. Thereversed phase method employs a C₁₈ (5 μm, 300 Å) column, stepwisegradient elution with mobile phase components consisting of 0.15%trifluoroacetic acid in water (solvent A) and 0.125% trifluoroaceticacid in acetonitrile (solvent B), and UV detection at 215 nm.

FIG. 12 shows representative full-scale and expanded-scale chromatogramsof a lyophilized reformulation of KINEVAC® upon reconstitution with 5 mLwater, resulting in a sincalide concentration of 1 μg/mL.

Other Embodiments

Although the present invention has been described with reference topreferred embodiments, one skilled in the art can easily ascertain itsessential characteristics, and without departing from the spirit andscope thereof can make various changes and, modifications of theinvention to adapt it to various usages and conditions. Those skilled inthe art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are encompassed by thescope of the present invention.

All publications, patents, and applications mentioned in thisspecification are herein incorporated by reference.

We claim:
 1. A stabilized, physiologically acceptable formulation ofsincalide comprising: an effective amount of sincalide, at least onestabilizer, at least one chelator, and at least one bulkingagent/tonicity adjuster.
 2. The formulation of claim 1, comprising about0.0008-0.0012 mg/mL sincalide.
 3. The formulation of claim 1, comprisingabout 0.005 mg/vial sincalide.
 4. The formulation of claim 1, whereinthe stabilizer is selected from the group consisting of antioxidants andamino acids.
 5. The formulation of claim 4, wherein the stabilizer is anantioxidant.
 6. The formulation of claim 4, wherein the stabilizer issodium metabisulfite.
 7. The formulation of claim 1, wherein theformulation comprises a plurality of stabilizers.
 8. The formulation ofclaim 7, wherein the stabilizers comprise L-arginine monohydrochloride,L-methionine, L-lysine monohydrochloride, or sodium metabisulfite. 9.The formulation of claim 7, wherein the stabilizers comprise L-argininemonohydrochloride and L-methionine.
 10. The formulation of claim 7,wherein the stabilizers comprise L-lysine monohydrochloride and sodiummetabisulfite.
 11. The formulation of claim 7, wherein the stabilizerscomprise L-arginine monohydrochloride and L-lysine monohydrochloride.12. The formulation of claim 7, wherein the stabilizers compriseL-methionine and sodium metabisulfite.
 13. The formulation of claim 7,wherein the stabilizers comprise L-arginine monohydrochloride,L-methionine, L-lysine monohydrochloride, and sodium metabisulfite. 14.The formulation of claim 8, comprising about 0.0008-0.0012 mg/mLsincalide.
 15. The formulation of claim 8, comprising about 0.005mg/vial sincalide.
 16. The formulation of claim 1, further comprising abuffer.
 17. The formulation of claim 1, wherein the bulkingagent/tonicity adjuster is selected from the group consisting ofmannitol, amino acids, lactose, potassium chloride, sodium chloride,maltose, sucrose, PEG's, trehalose, raffinose, dextrose, cyclodextrins,dextran, galacturonic acid, Ficoll, and polyvinylpyrrolidone (PVP). 18.The formulation of claim 17, wherein the bulking agent/tonicity adjusteris D-mannitol.
 19. A stabilized, physiologically acceptable formulationof sincalide comprising: about 0.005 mg/vial sincalide; at least onestabilizer wherein the stabilizers comprise L-argininemonohydrochloride, L-methionine, L-lysine monohydrochloride, or sodiummetabisulfite; at least one chelator; and at least one bulkingagent/tonicity adjuster selected from the group consisting of mannitol,amino acids, lactose, potassium chloride, sodium chloride, maltose,sucrose, PEG's, trehalose, raffinose, dextrose, cyclodextrins, dextran,galacturonic acid, Ficoll, and polyvinylpyrrolidone (PVP).
 20. Astabilized, physiologically acceptable formulation of sincalidecomprising: about 0.005 mg/vial sincalide; at least one stabilizer; atleast one chelator; and at least one bulking agent/tonicity adjusterselected from the group consisting of mannitol, amino acids, lactose,potassium chloride, sodium chloride, maltose, sucrose, PEG's, trehalose,raffinose, dextrose, cyclodextrins, dextran, galacturonic acid, Ficoll,and polyvinylpyrrolidone (PVP).